We are following two strategies to develop a live-attenuated pediatric RSV vaccine. (i) The primary strategy is to develop live-attenuated RSV strains, with attenuation provided mainly by deletion of one of several nonessential genes and by missense and codon-deletion mutations that are mainly in the L polymerase and have been stabilized against de-attenuation using reverse genetics. The immediate goal is to identify one or two lead candidates suitable for further development as an intranasal pediatric RSV vaccine. (ii) A secondary vaccine strategy is to use attenuated PIV strains (primarily PIV3 and secondarily PIV1) as vectors to express RSV antigen (primarily the fusion F protein) which provide live bivalent HPIV/RSV vaccines. The pre-clinical development of these PIV-vectored vaccines is described in the accompanying report Laboratory and Pre-Clinical Studies of Parainfluenza Viruses. Both vaccine strategies are being developed under a Cooperative Research and Development Agreement (CRADA) with Sanofi Pasteur, Inc. To date, our clinical trials have focused on live-attenuated RSV strains. We will evaluate a PIV-vectored vaccine in the clinic in 2020. One lineage of live-attenuated RSV vaccine candidates involves deletion of the ORF encoding the small (90 amino acids) viral M2-2 protein. The M2-2 protein plays a role in regulating RSV RNA synthesis, and its deletion results in down-regulated viral RNA replication (causing viral attenuation) and a global up-regulation of viral gene transcription and antigen synthesis. Increased antigen expression per genome raises the possibility of increased immunogenicity per infectious particle. Prototype delM2-2 candidates called RSV MEDI/delM2-2 and RSV LID/delM2-2 were evaluated and reported upon in past years. Based on these results, a number of derivatives were constructed and are in small Phase 1 pediatric clinical trials. These viruses are: RSV D46/cp/delM2-2 (ClinicalTrials.gov identifier NCT02601612), RSV LID/delM2-2/1030s (NCT02794870 and NCT0252339), RSV LID/cp/delM2-2 (NCT02890381 and NCT02948127), RSV D46/NS2/N/delM2-2 (NCT03099291 and NCT03102034), and RSV 276 (NCT03227029, NCT03422237, and NCT03916185). The relatively large number of candidates is because of incongruities in some of the results, making it necessary to make and evaluate a greater number of derivatives. This year, a report on a single delM2-2 candidate was published, namely RSV LID/cp/delM2-2 (NCT02890381 and NCT02948127). This virus is attenuated through deletion of M2-2 combined with five cold passage (cp) missense mutations present in the N, F, and L proteins. RSV-seronegative children ages 6-24 months received a single intranasal dose of 5.0 log10 plaque forming units (PFU) of LID/cp/delM2-2 (n=11) or placebo (n=6). RSV serum antibodies, vaccine infectivity, and reactogenicity were assessed. Vaccine virus was shed by 36% of vaccinees at low levels, and 45% had 4-fold rise in serum neutralizing antibodies. LID/cp/M2-2 was well-tolerated. However, infectivity and immunogenicity were lower than desired, suggesting over-attenuation. Therefore, the study was closed early, prior to full accrual. A second lineage of RSV vaccine candidates contains deletion of the NS2 gene, whose encoded protein antagonizes host interferon and apoptosis responses to viral infection. The candidate RSV delNS2/del1313/I1314L contains the delNS2 mutation combined with a mutation called del1313/I1314L comprising deletion of codon 1313 in the L polymerase plus an adjacent missense mutation I1314L that stabilizes against de-attenuation. This virus was evaluated in a Phase 1 pediatric clinical trial (NCT01893554), A single intranasal dose of RSV/NS2/1313/I1314L was administered at 6.0 log10 plaque-forming units (PFU) to 15 RSV-seropositive children ages 12-59 months, in whom the vaccine was very highly restricted. This indicated that this candidate was sufficiently attenuated to evaluate in seronegative infants and children 6-24 months of age. Therefore, the virus was administered at a dose of 5.0 log10 PFU to 15 vaccinees (with 6 placebo recipients). However, shedding was detected in only 11/15 (73%) recipients, and thus the vaccine was too attenuated at this dose. When given at a 10-fold higher dose (6.0 log10 PFU) to 20 vaccinees (with 10 placebo recipients), the vaccine was well-tolerated, 90% of recipients shed vaccine virus, 85% had a serum RSV-specific antibody response, and 100% were infected based on vaccine shedding and/or a serum antibody response. This vaccine is now considered to be a leading candidate, and is presently being evaluated in a Phase 1 study in a head-to-head comparison with a delM2-2 virus called RSV 276 (NCT03227029 and NCT03422237). A second delNS2-based vaccine candidate, RSV delNS2/1030s, combines the delNS2 mutation with a stabilized missense mutation called 1030s that consists of Y1321K and S1313(TCA) mutations in the L polymerase. This virus presently is being evaluated in a Phase 1 pediatric clinical study (NCT03387137). The 1030s mutation is somewhat less attenuating than the del1313/I1314L mutation, and therefore RSV delNS2/1030s should be less attenuated than RSV/NS2/1313/I1314L. The RSV delNS2/1030s, RSV/NS2/1313/I1314L, and RSV 276 candidates are presently being evaluated head-to-head in a Phase 1/2 clinical trial in seronegative infants and young children (NCT03916185). A third lineage of RSV vaccine candidates contains deletion of the NS1 gene that, like NS2, encodes a protein that antagonizes host interferon and apoptosis responses, but does so more efficiently than NS2 and thus might confer a phenotype that is more attenuated and immunogenic. Two viruses were made that each contain the delNS1 deletion as the sole attenuating element, but in one virus the F and G genes have been moved to the first and second genome positions in order to increase their expression (RSV 6120/delNS1 and 6120/F1G2/delNS1, respectively). These viruses presently are being compared head-to-head in a Phase 1 clinical trial (NCT03596801). The subjects in a number of Phase 1 studies were surveilled during the subsequent winter RSV season to measure serum RSV-neutralizing antibody titers at the beginning and end of the winter, and to monitor respiratory illness during the winter and identify causative agents by nasal wash and RT-PCR. Without going into detail, this surveillance has provided presumptive evidence of protection against wild-type RSV infection, as well as strong anamnestic RSV-specific antibody responses. These preliminary observations are very encouraging. cDNA-derived RSV strain A2 is presently being evaluated in an in-patient setting for infectivity, replication, pathogenesis, and immunogenicity in healthy adult volunteers in a dose-escalation study (NCT02484417). We also are evaluating a recent clinical isolate RSV A/Maryland/001/11 for which we have developed a reverse genetic system and recovered a recombinant virus that is presently in a Phase 1 trial in healthy adult volunteers (NCT03624790). These studies will provide an infection model that can be used to evaluate RSV therapeutic candidates and adult RSV vaccine candidates, and to study viral pathogenesis and the host response.